Biochemical and Biological Properties of Myosins

我们感兴趣的是i类和ii类肌凝蛋白的肌动蛋白激活的atp酶活性调控的分子基础，这些肌凝蛋白的生物学作用及其生物活性调控。在一个项目中，为了了解肌球蛋白头部和尾部对酶活性和功能的耦合贡献，我们研究了由Dictyostelium myosin II的运动结构域和棘阿米巴或平滑肌肌球蛋白II的尾部结构域组成的嵌合体的特性。嵌合体的肌动蛋白激活的atp酶活性比野生型盘基骨桥菌肌球蛋白II高10-15倍，但与野生型肌球蛋白相比，这种活性既不受调节轻链磷酸化的调节，也不受棘阿米巴嵌合体尾部磷酸化的调节。当在肌球蛋白ii缺失的细胞中表达时，两种嵌合体都恢复了肌球蛋白ii依赖的两种功能，即悬浮培养中的细胞分裂和con A受体的封顶，但不支持第三种肌球蛋白ii依赖的功能，即向子实体的完全发育。之前已经有研究表明，Dictyostelium myosin II的尾部结构域包含了定位到分裂细胞的卵裂沟（cleavage row， CF）所需的所有信息。我们现在发现，在肌球蛋白II缺失的细胞中表达时，283个残基组装域（体外组装的最短尾部片段）进入CF，棘阿米巴肌球蛋白II尾部的256个残基区域也是如此。因此，肌球蛋白定位到CF似乎没有特定的序列要求。我们比较了15个Dictyostelium myosin II的HCM/ ted -site表面环突变体的能力，以支持三种myosin-II依赖的功能。鸡平滑肌肌球蛋白环支持正常的con - a受体封顶，80%的正常生长，只有轻微的发育受损。人β -心肌肌球蛋白环仅支持野生型25%的生长速度，不完全封顶和严重受损的发育，即到尖端丘阶段，仅略好于肌球蛋白II-null细胞。各种点突变使平滑肌环充分发挥功能，使心脏环的功能更像平滑肌环。研究人员正在研究所有突变体的肌动蛋白激活的atp酶和体外运动活性，以确定如果有的话，哪一种与它们的生物学特性相关。棘阿米巴和曲霉肌球蛋白I重链头、颈、尾结构域和轻链以不同组合互换的嵌合体研究进一步证明了肌球蛋白活性的三个重链结构域和轻链的相互依赖性。

We are interested in the molecular bases of the regulation of the actin-activated ATPase activities of class-I and class-II myosins, the biological roles of these myosins and the regulation of their biological activities. In one project, to understand the coupled contributions of myosin heads and tails to enzymatic activity and function, we studied the properties of chimeras consisting of the motor domain of Dictyostelium myosin II and the tail domains of either Acanthamoeba or smooth muscle myosin II. The chimeras have 10-15 higher actin-activated ATPase activity than wild-type Dictyostelium myosin II but, in contrast to wild-type myosins, this activity is not regulated by phosphorylation of either the regulatory light chain or, in the Acanthamoeba chimera, phosphorylation of the tail. When expressed in myosin II-null cells, both chimeras rescue two myosin II-dependent functions, cytokinesis in suspension culture and capping of con A receptors, but do not support a third myosin II-dependent function, full development to fruiting bodies. It had previously been shown by others that the tail domain of Dictyostelium myosin II contains all the information needed for localization to the cleavage furrow (CF) of dividing cells. We have now found that the 283-residue assembly domain, the shortest tail segment that assembles in vitro, goes to the CF when expressed in myosin II-null cells, as does also an equivalent 256-residue region from the tail of Acanthamoeba myosin II. Thus, there seems to be no specific sequence requirement for localization of myosin to the CF. We have compared the abilities of 15 mutants of the HCM/TEDS-site surface loop of Dictyostelium myosin II to support the three myosin-II dependent functions. The chicken smooth muscle myosin loop supports normal con A-receptor capping, 80% of normal growth and only slightly impaired development. The human beta-cardiac myosin loop supports growth at only 25% of wild-type rate, incomplete capping and greatly impaired development, i.e. to the the tipped mound stage, only slightly better than myosin II-null cells. Various point mutations make the smooth muscle loop fully functional and the cardiac loop function more like the smooth muscle loop. The actin-activated ATPase and in vitro motility activities of all of the mutants are being studied to see which, if either, correlates with their biological properties. Studies of chimeras in which the heavy chain head, neck and tail domains and light chains of Acanthamoeba and Aspergillus myosin I are interchanged in various combinations provide further evidence for the interdependence of the three heavy chain domains and light chains for myosin activities.